Complexes of the G protein ?subunit G?5 with the R7 family of RGS proteins serve as GAPs for Gi family and are involved in many physiological functions. Both G?5 and R7 proteins were originally found in the nervous system, but recently detected in some other tissues, notably endocrine glands. The current study concentrates on the role of G?5-R7 in pancreatic ? cells and advances an original line of research developed in this laboratory: regulation of M3 muscarinic receptor (M3R) by G?5-R7. The most recent study identified a novel pathway where G?5-R7 can potentiate M3R-induced Ca2+ influx across the plasma membrane. This effect on Ca2+ flux is consistent with the higher level of insulin secretion in the presence of G?5-R7. The positive effect is novel and exciting because RGS proteins are thought to be negative regulators of G protein signaling. Another puzzling detail is that M3R is a Gq-coupled receptor, and G?5-R7 members do not have GAP activity for Gq. The project will pursue three well-integrated aims. To determine specific ? cell signaling mechanisms sensitive to G?5-R7, Aim 1 will target members of the M3R-to-insulin pathway with pharmacologic inhibitors or gene knockout and knockdown. Studies will be performed in pancreatic islets from WT and G 5 knockout mice and insulinoma cell line Min6. Experiments will measure insulin secretion, [Ca I] and other signaling responses to 2+ M3R agonists. Aim 2 will generate and analyze mice with ? cell-specific knockout of G?5. Primary islets from these mice will be examined by signaling and secretion assays in vitro and long-term functionality of these islets will be studied after their intraocular transplantation into mice with induced diabete. Aim 3 will determine the effect of G?5-R7 complex on gene expression using a combination of RT-PCR, in situ hybridization and next generation sequencing of pancreatic islet RNA from G?5 KO mice. These studies will have a strong impact on understanding pancreatic ? cell signaling, insulin secretion and adaptation to stress. The new mechanistic insights will provide a sustained flow of ideas for the broad area of GPCR signaling, regulation of hormone release and etiology such of metabolic disorders as diabetes and obesity.